Effect Of Microglial Activation On Thalamic Hemorrhage-induced Pain

BackgroundCentral post stroke pain(CPSP)is a kind of chronic neuropathic pain directly related to the focus of stroke,which often appears continuously or intermittently.Because of the intense pain produced by CPSP,the nature of pain is difficult to describe.Besides,clinicians have little knowledge of CPSP,so that patients are in a state of pain for a long time.After that,the emotional changes such as depression and anxiety aggravate the pain of patients,which lead to a vicious circle.It is reported that about 1-14%of stroke patients around the world have CPSP within a few months of onset,and thalamic hemorrhage is the main cause of CPSP.After thalamus hemorrhage,patients have symptoms of pain,abnormal mood,learning and memory disorders,which seriously affect the quality of life and social functions of patients.Therefore,it is very important to study the pathogenesis of CPSP to improve the prognosis of thalamic hemorrhage.Not only the primary hemorrhage,but also the secondary injury plays an important role in the production and development of CPSP after thalamic hemorrhage.As an important part of secondary injury,neuroinflammation induces glial cell over activation and neuronal injury,which leads to neuropathic pain.Microglia are macrophages in the brain,which are innate immune cells in the central nervous system.They can act as the first responder of nerve injury and play an extremely important role in the physiological process of the central nervous system.In previous studies,we found that the microglia changed the physiology and morphology during the central neuropathic pain models.Besides,a variety of receptor activation and pro-inflammatory factors which were released on the cell surface can participate in the production of pain.Inhibition of microglia and activation of intracellular signaling pathway can slow down the neuropathic pain.In conclusion,the activation of microglia may lead to the production of CPSP.Therefore,in this study,microglia will be depleted to observe whether microglia can relieve pain.And we will further clarify the role of microglia in the development of CPSP.The survival of microglia depends on the signal transduction of colony-stimulating factor-1 receptor(CSF1R).Colony-stimulating factor-1 is a key cytokine involved in the recruitment and activation of microglia.Inhibition of CSF1R can depletion microglia in the central nervous system rapidly.PLX3397 is an effective CSF1R inhibitor,which can freely cross the blood-brain barrier and combine with high affinity CSF1R to competitively inhibit the recruitment and activation of microglia in the brain.It was found that PLX3397 could depletion 90%of microglia in the brain for 21 days without affecting the expression of other intrinsic cells,including neurons,astrocytes or oligodendrocytes.Therefore,PLX3397,a specific microglia inhibitor,was selected to verify the role of microglia in the production of CPSP.Toll like receptor 4(TLR4)is a key innate immune receptor,which is mainly expressed in microglia in central nervous system and can be used as a molecular target to activate microglia.It has been reported that oxidative stress can raise the expression of TLR4 in human macrophages and activate Nuclear factor kB(NF-?B)signaling pathway in order to stimulate the release of downstream inflammatory factors.In addition,TLR4 can also participate in innate,adaptive immune responses,the induction,transformation and maintenance of chronic pain and neuropathic pain.Previous studies have found that TLR4 gene knockout(KO/TLR4-/-)mice can inhibit brain damage caused by cerebral hemorrhage,reducing brain edema and neurological damage,and reduce cell apoptosis compared with wild-type mice.However,whether TLR4 mediates the activation of microglia in the CPSP model has not been reported.In order to verify whether TLR4 is involved in the production of CPSP by mediating the activation of microglia,TLR4 gene knockout mice were selected to observe the activation of microglia and the change of the pain threshold.And then,we study the molecular mechanism of TLR4 involved in the production of CPSP more deeply.In this paper,we found that small glue cells participate in the production of CPSP through the study of the pathogenesis of CPSP.The aim of this study is to provide theoretical and experimental basis for the treatment of central pain after stroke.Purpose(1)Establish CPSP model of thalamic hemorrhage in mice,and detect the pain threshold or other related behavioral changes.Observe the morphological changes of myelin sheath,dead neurons and glial cell activation around the hematoma,andresearch whether the activation of microglia and its surface receptor TLR4 mediates the generation of neuritis.(2)PLX3397 was used to remove microglia from the brain of CPSP model mice,and the changes of mechanical pain threshold,cold pain threshold,microglia activation and TLR4 signal pathway protein expression were detected after operation.Whethermicroglia mediated neuroinflammation through TLR4 Signal pathway activation or participated in the development of CPSP.(3)After the TLR4-/-mice were used to establish the model,the pain threshold and immunofluorescence detection confirmed that TLR4 Protein participated in the over activation of microglia and induced CPSP.Methods(1)To establish a model of thalamic hemorrhage in mice to detect the pathological changes and evaluate the success of CPSP model by behavioral analysis.The mice were divided into Sham group and CPSP group.The model of thalamic hemorrhage was established by stereotaxic injection of type VII collagenase.After modeling,the location and volume of hematoma were detected by D1 fresh brain section,the number of myelin sheath around hematoma was detected by Crystal violet staining solution/Fast blue(CV/LFB)double staining and the number of dead neurons around hematoma was detected by Fluoro-jade B(FJB)staining.Behavioral tests were performed at different time points after modeling,including motor function test(neurological function score and corner test),pain threshold test(mechanical pain threshold and cold pain threshold),anxiety and depression like emotion test(open field,elevated plus-maze,forced swimming,tail suspension)and learning and memory test(Y-maze,novel object recognition),to evaluate whether the CPSP model was successfully established.(2)Study on the pathogenesis of CPSP.At D3 after thalamic hemorrhage,immunofluorescence staining was used to detect the activation of microglia and astrocytes around hematoma and the location of TLR4 Protein.(3)Clarify the role of microglia in the development of CPSP after intracerebral hemorrhage.At D21 before the establishment of the model,normal C57BL/6J mice were given PLX3397(concentration:10mg/ml,dose of each mouse:40mg/kg)by oral gavage once a day until the materials were taken after the operation.The mice were randomly divided into two groups:CPSP+vehicle group and CPSP+PLX3397 group.The changes of pain threshold was detected by D0,D1,D3,D5 and D7,hematoma size,myelin sheath around hematoma,number of dead neurons and activation of glial cells were detected by D3,respectively,and TLR4/MyD88 signal pathway protein expression was detected by D7 WB.(4)Analyze the signal pathway of microglia involved in the development of CPSP induced by neuroinflammation.TLR4-/-mice were used to establish the model.The experimental animals were divided into three groups:WT Sham group,WT CPSP group and KO CPSP group.The changes of pain threshold of mechanical pain threshold and cold pain threshold were detected by D0,D1,D3,D5 and D7,and the activation of glial cells around hematoma was detected by D3 morphological staining.Results(1)D1 hematoma appeared after thalamic hemorrhage,the number of myelin sheaths around D3 hematoma decreased,and the number of death neurons increased.After thalamic hemorrhage,the mice had no motor impairment,but the postoperative cold pain threshold of D1 mice reduced and continued to D14,and the postoperative mechanical pain threshold of D3 continued to D14(p<0.05).Anxiety,depression and learning and memory dysfunction also occur in D14 mice after CPSP(p<0.05),suggesting that CPSP modeling was successful.(2)After CPSP modeling D3,microglia and astrocytes around hematoma were activated in large numbers(p<0.05),and TLR4 protein was mainly expressed on the surface of microglia.(3)After using PLX3397,the pain threshold of D1 cold pain threshold was significantly reduced,and the pain threshold of D3 mechanical pain threshold was significantly improved(p<0.05);the number of myelin sheath around hematoma increased,the number of dead neurons and microglia activation decreased significantly(p<0.05);the protein expression of TLR4/MyD88 signal pathway decreased significantly(p<0.05).(4)After using TLR4-/-mice to establish CPSP models,the mechanical pain threshold and cold pain threshold were relieved significantly(p<0.05),and the number of glial cell activation was decreased significantly(p<0.05).Conclusions(1)After injecting collagenase to establish the thalamic hemorrhage model,microglia were over activated,and the release of pro-inflammatory cytokines led to the development of CPSP.(2)The activation of TLR4 on the surface of microglia and its downstream signaling pathway mediates the occurrence and development of CPSP.